In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L9(33) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5 L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71 μg/ml in the shake-flask, and 613.47 μg/ml in the 5 L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280 rpm for 4 days at 26 °C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.
Keywords: Biological activity; Ganoderma lucidum; fermentation parameters; fungal immunomodulatory protein; orthogonal experiment.